Microorganisms generally live attached to surfaces in many natural, industrial, and medical environments, encapsulated by extracellular substances including biopolymers and macromolecules. The resulting layer of slime encapsulated microorganism is termed a biofilm. Biofilms are the predominant mode of growth of bacteria in the natural environment, and bacteria growing in biofilms exhibit distinct physiological properties. Compared to their planktonically grown counterparts, the bacteria in a biofilm are more resistant to antibiotics, UV irradiation, detergents and the host immune response (Gristina et al. 1988. Journal of the American Medical Association, 259: 870-874; Stewart. 1994. Antimicrobial Agents & Chemotherapy, 38(5): 1052-1058; Costerton et al. 1995. Annu. Rev. Microbiol., 49: 711-745; Maira-Litran et al. 2000. Journal of Applied Microbiology, 88: 243-247). A biofilm may include one or more microorganisms, including gram-positive and gram-negative bacteria, algae, protozoa, and/or yeast or filamentous fungi and viruses and/or bacteriophage. Examples of problematic biofilms are dental plaque, infections on medical implants, but also the initial fouling on ship hulls (Satuito et al. 1997. Hydrobiologia, 358: 275-280). Biofilms are attributed to the pathogenesis of many infections in humans and are a significant problem in industry in terms of biofouling of exposed surfaces where biofilm colonisation can form the base component of a localised ecosystem which can disrupt and interfere with industrial processes and components. New strategies are required to inhibit biofilm formation, disperse existing biofilm and to trigger bacteria in a biofilm to return to the antibiotic-sensitive planktonic state.
Many types of microbes grow naturally in a biofilm context, such as bacteria, fungi, algae etc.
It is known in the art that biofilms can have, as a component, DNA (termed extracellular DNA or eDNA) although its function there, remains unknown. Certain groups have sought to employ nuclease enzymes to disrupt biofilms. However, the prior art use of nucleases in this respect has been limited to human DNase and DNase I, an enzyme purified from bovine pancreas and sold commercially.
For instance, WO 06/017816 discloses compositions and methods for the inhibition of biofilm formation or reduction of existing or developing biofilms in a patient. The methods include administering to a subject that has or is at risk of developing biofilms a compound or formulation that inhibits the formation or polymerization of actin microfilaments or depolymerizes actin microfilaments at or proximal to the site of biofilm formation. Such a compound can be administered in combination with a compound or formulation that inhibits the accumulation or activity of cells that are likely to undergo necrosis at or proximal to the site of biofilm formation (i.e., neutrophils). The methods and compositions can further include the use of anti-DNA and/or anti-mucin compounds.
WO 2009/121183 discloses an anti-biofilm composition comprising two or more agents selected from the group consisting of DISPERSIN B, 5-Fluorouracil, Deoxyribonuclease I (bovine DNase I) and Proteinase K for preventing growth and proliferation of biofilm-embedded microorganisms.
Prior art studies have demonstrated that bovine DNase I both prevented biofilm formation and (to a certain extent) dissolved existing biofilm colonies. It was concluded that extracellular DNA is required for the initial establishment of P. aeruginosa biofilms, which is later strengthened by other substances such as exopolysaccharides and proteins (Whitchurch et al. 2002. Science, 295: 1487).
Purified recombinant human DNase1L2 was shown to suppress biofilm formation by Pseudomonas aeruginosa and Staphylococcus aureus (Eckhart et al. 2007. British Journal of Dermatology, 156(6): 1342-1345; Tetz et al. 2009. Antimicrobial Agents & Chemotherapy, 53(3): 1204-1209).
It has been shown that type IV pilli of Pseudomonas aeruginosa bind DNA, and that this function is conserved throughout the type IV pilli in bacteria (van Schaik et al. 2005. Journal of Bacteriology, 187(4) 1455-1464). Both DNA and type IV pilli are involved in the attachment to a surface, the initial stage of biofilm formation.
Furthermore, it has recently been described that in single species biofilms of Bacillus cereus or the marine photosynthetic bacterium Rhodovulum sp. not only DNA but also RNA is present in the extracellular matrix (Vilain et al. 2009. Applied and Environmental Microbiology, 75(9): 2861-2868; Ando et al. 2006. Journal of Biochemistry, 139: 805-811).
In view of the above, it is clear that DNA and RNA are structural components of biofilm and that alternative, more effective nuclease enzymes, other than bovine DNase 1 would be beneficial in terms of biofilm disruption and prevention in a range of applications, including medical and non-medical anti-biofouling applications.
In particular, the removal of biofilms in medical contexts currently poses significant problems since the bacteria present in the biofilm are highly resistant to many antimicrobial compounds. Furthermore, prior art methods for biofilm disruption involve compositions active against mainly only gram negative proteobacteria, and show very specific activity against a limited number of strains. This significantly limits their utility. Thus, there remains a need for new biofilm disruption and prevention methods and strategies involving compositions with improved properties.